IHC image of ab54362 staining in human T-cell lymphoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab54362, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-CBLB antibody [246C5a] (ab54362) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CBLB knockout HeLa cell lysate
Lane 3 : CTLL-2 cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 109 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab54362 observed at 130 kDa. Red - loading control, ab181602 observed at 37 kDa.

ab54362 Anti-CBLB antibody [246C5a] was shown to specifically react with CBLB in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264711 (knockout cell lysate ab258342) was used. Wild-type and CBLB knockout samples were subjected to SDS-PAGE. ab54362 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

Anti-CBLB antibody [246C5a] (ab54362) + immunizing recombinant protein

Predicted band size: 109 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?



The molecular weight of the band on the Western blot does not correspond to the molecular weight of the natural protein as the immunizing recombinant protein fragment was used as the test antigen.